Progress 07/01/23 to 06/30/24

Outputs
Target Audience:We conducted a Customer Discoveryto identify potential customers for EmergingDx's diagnostic aquaculture solution. In addition to identifying the companies, with our TABA provider researched key points of contact at each company and provided contact information that includes name, title, email address, phone number, and location. We had interviews with two companies to understand thier needs and explore the interest to our solution. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Oral presentations given at professional meetings and conferences in which this ongoing research was described: 1) Lennox SMG, Shavalier MA, Brenden TO, Knupp CK, Call DR, Soto E, Zhang Q, Loch TP. Developing a rapid diagnostic assay to prevent bacterial coldwater disease vertical transmission. Oral presentation given at the Michigan Department of Natural Resources Fish Production Section Meeting, Sault St Marie, MI, 2024. 2) Loch TP. Collaborative efforts to enhance aquatic animal health in the Great Lakes basin and beyond. Promotion and Tenure Seminar, Dept. of Fisheries & Wildlife and Dept. of Pathobiology & Diagnostic Investigation, Michigan State University, East Lansing, MI, 2023. 3) Knupp CK, Lennox SMG, Call D, Soto E, Shavalier MA, Shrestha N, Faisal M, & Loch TP. Flavobacteria: An Emerging and Resurging Threat to Fish Health Worldwide. Invited Seminar for the Egyptian Aquatic Health Association Monthly Seminar series, Cairo, Egypt, 2023. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we will perform the following tasks: 1) Test and optimize our LAMP Microfludic chip to determinethe limits of detection (LOD), linear output ranges, and dynamic ranges of the LAMP assay via varying concentrations of the lysed pathogen in buffer. 2) Test the LAMP chip againist biological samples toevaluate robustness to potential interferents in complex samples and provide an initial demonstration of the field-forwardness of our assay, we will test our optimized LAMP assay against simulated biological samples. 3) Perform the preliminary cost analysis, construct a bill of materials (BOM), and initial concept of operations (CONOPS) for the LAMP microfluidic assay.

Impacts
What was accomplished under these goals? Flavobacterium psychrophilum is not the only fish pathogen that heavily relies on vertical transmission to perpetuate infections. Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD), is also efficiently transmitted from infected broodstock to their offspring through contaminated eggs. Both pathogens pose significant challenges to salmonid farming, such as the lack of effective vaccines, inconsistent egg disinfection methods, and increasing antimicrobial resistance. However, there have been notable successes in managing BKD, particularly through testing-based segregation and culling of infected eggs. For instance, it has been demonstrated that testing kidney tissues of terminally spawning salmon for R. salmoninarum using enzyme-linked immunosorbent assay (ELISA) and culling the eggs from infected broodstock could substantially reduce the prevalence and severity of BKD in subsequent generations. This method, along with enhanced biosecurity, has also been used to significantly lower R. salmoninarum infection rates in hatchery-reared Pacific salmon in the Great Lakes. A key difference between the two pathogens is that R. salmoninarum is a relatively homogenous bacterium, while F. psychrophilum presents significant challenges in detection due to its genetic diversity. Our research has revealed limitations with the few serological reagents available for detecting F. psychrophilum. Given the demonstrated success of testing-based culling in controlling BKD, and the strong evidence that vertical transmission plays a critical role in F. psychrophilum-induced BCWD losses in U.S. salmonid farms, we argue that there is an urgent need for a rapid, sensitive, and field-deployable test for F. psychrophilum to help curb its vertical transmission. EmergingDx's LAMP diagnostic technology would have immediate commercial and government applications for the screening of transboundary or nationally reportable pathogens in domestic animals in agro-industrial and aquaculture settings. We envision the immediate demand would come from Federal Government agencies, including the DHS, USDA, HHS, and state, local, tribal, and territorial governments, for early detection of potential outbreaks within agro-industrial settings, screening food products for contaminants that pose a risk to public health, and allowing the TSA, Customs and Border Protection, and USDA inspectors to intercept either infected animals or contaminated food products at points of entry. Farmers, ranchers, and food production companies would also be likely to use such a device to monitor their livestock, animal feed, or products to prevent the spread of infections or sale of harmful products to the public. During this reporting period, we successfully developed several disposable microfluidic devices optimized for our LAMP (Loop-Mediated Isothermal Amplification) assays. Microfluidic systems offer a multitude of advantages, including minimized sample volumes, shorter processing times, reduced reagent consumption, and overall cost-efficiency, making them ideal for applications in diagnostics. The integration of biosensors with microfluidics further amplifies these benefits, enabling laminar flow control, minimal handling of hazardous materials, simultaneous multi-sample processing, portability, and design versatility. These attributes are especially important in point-of-care (POC) diagnostics, where rapid, accurate, and accessible testing is essential. We focused on microfluidic chip design by meticulously defining parameters such as channel dimensions, chamber geometries, and system configurations to align with the unique requirements of LAMP assays. The developed microfluidic device incorporates multiple critical components, including inlets for nuclease-free water and buffer solutions, a sample lysis chamber, an inline passive mixer, and incubation/detection chambers--designed either as single or multiple units, depending on the specific assay. Our initial prototype microfluidic device was fabricated from PDMS (polydimethylsiloxane) and sealed with a glass substrate to maintain optimal structural integrity and fluid dynamics. Flow behavior within the microfluidic channels was rigorously evaluated under various conditions essential for LAMP assays, ensuring the system's reliability. The device effectively integrates two key processes: (1) sample preparation via cell lysis and (2) colorimetric detection through LAMP assay incubation. This combination streamlines the diagnostic workflow and enhances the overall analytical capability of the device. To complement the microfluidic system, we designed and built a handheld, reusable heater equipped with precision electronics. This heating system is based on a PCB-integrated design, featuring two resistive heaters (heater 1 and heater 2) constructed with tightly-routed metal layers. When an electric current is passed through these layers, the heaters produce uniform heating. Temperature regulation is achieved via a sensor IC with a range of 0-150°C, offering a sensitivity of approximately 0.5°C. The sensors are strategically placed directly beneath the center of each heating pad, allowing for accurate surface temperature readings without significant time delays. The sensor's sensitivity, rated at 10 mV/°C, coupled with the system's analog-to-digital converter (ADC) module, ensures high-resolution temperature measurement with an accuracy of ±0.5°C. For precise temperature control, a PID (Proportional-Integral-Derivative) controller is employed, maintaining stable temperature profiles despite external disturbances such as ambient temperature fluctuations or airflow. The closed-loop control firmware, running on a microcontroller, samples the temperature at a rate of 100 ms, which is more than sufficient given the heater's natural time constant of approximately 5-10 seconds. This enables the system to respond quickly to changes, ensuring that desired temperature profiles are achieved efficiently. At present, manual PID tuning allows us to reach steady-state temperature accuracies of ±1°C for heater 2 (maximum temperature: 75°C) and ±1.5°C for heater 1 (maximum temperature: 115°C). Our testing of the heaters demonstrated that heater 1 maintains the lysis chamber at 100°C, while heater 2 reliably sustains the incubation chamber at 63°C, both critical for optimal LAMP assay performance. In a significant milestone, we successfully confirmed the functionality of our LAMP master mix reagents following lyophilization and subsequent rehydration. This achievement ensures long-term stability of the reagents, a key factor for POC applications, especially in resource-limited settings. We performed comparison experiments for our LAMP Assay with Dual-Priming Isothermal Amplification (DAMP) Assay in batch mode. Briefly, to test the performance of our originally developed F. psychrophilum loop-mediated isothermal amplification (LAMP) assay, the LAMP primers were modified and adapted for dual-priming isothermal amplification (DAMP), a novel isothermal amplification method designed to reduce non-specific amplification, following the primer design parameters described the literature and targeting the same region of the F. psychrophilum gyrB gene. The LAMP assay was then compared to the newly devised DAMP assay. The results showed that our LAMP assay outperformed the DAMP assay both in terms of time to results and resilience to nonspecific amplification when run simultaneously and in duplicate. No apparent non-specific amplification occurred in the LAMP assay after an hour of incubation, further supporting the field-applicability of the assay. Finally, this integrated microfluidic system, combined with the reusable heating solution, offers a robust platform for LAMP assays, advancing the potential for efficient, reliable, and portable diagnostic tools.

Publications